polymers
Review
Milk Protein Polymer and Its Application
in Environmentally Safe Adhesives
Mingruo Guo 1,2, * and Guorong Wang 2
1
2
*
College of Food Science, Northeast Agricultural University, Harbin 150030, China
Department of Nutrition and Food Sciences, The University of Vermont, Burlington, VT 05405, USA;
[email protected]
Correspondence: [email protected]; Tel.: +1-802-656-8168
Academic Editor: Antonio Pizzi
Received: 26 June 2016; Accepted: 23 August 2016; Published: 31 August 2016
Abstract: Milk proteins (caseins and whey proteins) are important protein sources for human
nutrition; in addition, they possess important natural polymers. These protein molecules can be
modified by physical, chemical, and/or enzymatic means. Casein is one of the oldest natural
polymers, used for adhesives, dating back to thousands years ago. Research on milk-protein-based
adhesives is still ongoing. This article deals with the chemistry and structure of milk protein
polymers, and examples of uses in environmentally-safe adhesives. These are promising routes
in the exploration of the broad application of milk proteins.
Keywords: milk protein; casein; whey; glue; non-food; polymer
1. Introduction
Bovine milk contains about 3.2% protein, 3.4% fat, and 4.8% lactose, as well as various minerals
and vitamins. It is an important food for most people around the world. Due to its high nutritional
value, milk and its components are widely used in foods in various forms. Additionally, non-food
applications are also a large part of milk utilization, dating back to the ancient Egyptians in their use
of casein as glue [1] till today’s applications, such as in environmentally-safe wood adhesives and
paper/label adhesives [2–4].
Milk proteins are categorized as caseins and whey proteins, based on different solubilities at a
pH of 4.6 [5], which are approx. 2.6% and 0.6%, respectively, in bovine milk. The properties of casein
and whey protein (such as the chemistry, structure, functionality, nutrition, etc.) have been extensively
studied over the past few centuries. Casein exists in fresh milk in the form of a “micelle” structure,
which is a complex aggregate of proteins (α-, β-, and κ-casein) and colloidal phosphate calcium
(CCP) [6]. Whey proteins are a group of globular proteins, which consist mainly of β-lactoglobulin
(β-Lg), α-lactalbumin (α-La), and bovine serum albumin (BSA). Both caseins and whey proteins exhibit
unique polymer properties [7–9].
As with many other naturally-produced polymers, such as starch, tree gums, and clays, milk
protein components exhibit excellent adhesive properties, and have been used as one of the major
natural adhesive ingredients for thousands years, until the advent synthetic petroleum-based
polymers [10]. Natural adhesives are safe, renewable, and environmentally safe; in addition, they
possess excellent bonding properties. One of the reasons why caseins or natural polymers have been
steadily replaced by synthetic polymers is the cost [2], as milk protein is a food of high nutritional value,
and the demands for milk protein for use in food has been increasing [10]. However, the concepts
of natural and environmentally-safe adhesives are gaining more and more public attentions. Events
where children eat glue/paste, or where people get sick from formaldehyde emissions, have been
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reported. Natural and safe polymers should be reconsidered for use as adhesives in situations such as
office and school paper glues, wood adhesives, and any other high-value-added adhesives.
Due to the development of modern technology, the chemistry, structure, and functionality of milk
proteins have been extensively studied. This new knowledge is helpful in the formulation of more
efficient and safe adhesives. The objective of this article is to review and discuss the application of
milk protein in the formulations of environmentally-safe adhesives, and summarize current and past
examples of milk-protein-based adhesives.
2. Physicochemical Properties of Milk Proteins
Milk proteins are a heterogeneous mixture of proteins, and constitute approximately 3.0%–3.5%
of bovine milk. About 80% of bovine milk protein is called casein, which precipitates at pH 4.6.
The protein remains that are soluble in milk serum at pH 4.6 are called whey protein. The typical
compositions of milk proteins are listed in Table 1. In general, casein and whey proteins have very
different properties. Casein is mostly a random coil with a high proline content, highly phosphorylated
for calcium binding, and exists in large casein micelles; whey protein has ordered secondary structures
with a low proline content, is non-phosphorylated, and has small soluble proteins. In addition, casein
is sensitive to acid and is stable when heated, while whey protein is stable in acid and is sensitive
to heat.
Table 1. Major proteins in bovine milk.
Proteins
Content, g/L
Casein
α-Casein
β-Casein
κ-Casein
Minor constituents
Whey Protein
β-lactoglobulin
α-lactoalbumin
Serum albumin
Minor constituents
25
12
9
3.25
0.75
5.4
2.70
1.20
0.65
0.85
2.1. Caseins
Due to the high level of proline residue, casein molecules are able to form α-helix and β-sheet,
which are essential factors for secondary protein structures [11]. About 85% to 90% of casein
in bovine milk exists in a colloidal form, known as micelles [12,13], which are porous, spherical
aggregates with diameters ranging from 50 to 600 nm [14,15]. Various models of casein structure
have been suggested [6,9,12,14,16,17]. It has been suggested that the integrity of the micelle is
attributed to the hydrophobic interactions between casein molecules [18], between caseins and calcium
phosphate nano-clusters [19], or between polymeric casein aggregates and casein-calcium phosphate
aggregates [20]. The structure of the casein micelle is still elusive, but the “hairy” layers of κ-casein
on the surface of the micellar structure are generally accepted [21,22]. The casein micelle is extremely
stable in heat treatment, due to the κ-casein layers [12]. κ-casein is the only glycosylated casein with
oligosaccharides attached to throline residues in the C-terminal, and the only calcium insensitive
casein [23]. The C-terminal, due to the increased hydrophilicity, is the “hairy” structure that extends
into the serum solution, which contributes a stabilizing force [23,24]. During the production of cheese
or rennet casein, chymosin is added to cut off the κ-casein tail, and the casein collapses to form a weak
gel due to the loss of the inter-micellar repulsion force [22]. That is why sweet whey protein (the whey
produced from cheese making) contains a significant amount of Glycomacropeptide (GMP), which is
one third of κ-casein (residues 106–169) and is present in whey protein products [25]. The casein micelle
is made up of 94% casein protein and 6% CCP [26]. CCP stabilizes the casein micelle by crosslinking
Polymers 2016, 8, 324
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casein aggregates [27–30]. During acidification, such as using acid casein, in yogurt making, or in the
natural milk-souring process, CCP is dissolved when the pH drops and causes a gradual dissolution
of micellar integrity [26,28], and then casein starts to aggregate or precipitate.
The casein micelle is a very complex polymer aggregate, and interacts via hydrophobic and
electrostatic interactions and calcium bridging [31]. A typical casein micelle contains about 10,000
casein molecules [11]. In the past, bovine casein molecule were usually called random coil protein
because of the high content of proline residues, which prevents the formation of α-helix or β-sheet
structures [32,33]. Casein has extreme heat stability and shows no evidence of denaturation to a more
disordered structure [34]. The term “rheomorphic” was also suggested for this type of protein [35].
Caseins, considered rheomorphic proteins, have been adopted by many researchers [36–38]. According
to de Kruif and Holt’s definition, a rhomorphic protein is “one with an open conformation and therefore
has a considerable degree of side chain, and possibly also backbone, conformational flexibility” [5].
2.2. Whey Proteins
Whey is the liquid that remains after milk is curdled or strained. In the dairy industry, whey
refers to the co-products during the process of cheese, casein, or yogurt manufacturing. Cheese whey
is currently the most abundant commercially available whey product. Whey proteins are a group of
soluble proteins that are present in the milk serum phase at a pH of 4.6.
β-Lg is the dominant protein in whey, which accounts for about 50% of total whey proteins.
The molecule is comprised of 15% α-helix (residues 129–143, 65–76 ), 50% β-sheet (residues 1–6, 11–16,
39–45, 80–85, 92–96, 101–107, 117–123, and 145–151), and 15%–20% reverse turn (residues 7–10, 49–52,
61–64, 88–91, and 112–115) [39]. It has a very organized secondary structure, which is described as
a “cup” or “calyx” shaped three-dimensional structure [40,41]. Each monomer has one thiol group
(residue 121), and two disulphide bonds (residues 160–66 and 106–119) [42–44]. The presence of the
thiol group and the disulphide bond gives β-Lg an excellent gelation property via a thiol-disulphide
interchange under heat treatment [45]. β-Lg can exist in the form of a monomer, dimer, or octamer via
the disulphide bonds, depending on pH, temperature, and ion strength [46,47]. It exists as a dimer in
neutral pH aqueous solutions [47].
α-La is a globular protein with a two-lube structure [48]. It has 123 amino acid residues and
contains four disulphide bonds, but has no thiol group [8,49]. The absence of the thiol group gives
α-La a better heat stability than β-Lg. Unlike the thermo-gelling property of β-Lg, α-La forms a molten
globule state under mild denaturing conditions [7,50].
BSA is the third major proteins in whey. It is found in both mammal blood and milk. The molecular
structure of BSA is much large than that of β-Lg or α-La; it has approximately 580 residues. BSA has
both thiol groups and disulphide bonds [51].
3. Milk Protein Products
3.1. Casein Products
Casein products include rennet casein, acid casein, and caseinate [52]. Production starts with skim
milk, and then casein is separated from milk serum by adding rennet (mimicking cheese making), acid
(mimicking natural milk souring) [53], or using membrane filtration [54]. Rennet is an enzyme complex
that comes from the stomach of ruminant mammals. Chymosin is the key functional enzyme, which
hydrolyzes the bond between methionine and phenyalanine in κ-casein; thus, the casein coagulated
into three-dimensional networks. The gel is cut, stirred, drained, and washed before being dried into a
powder [55]. Casein can be precipitated at the pH of its isoelectric point, which is thought to be 4.6.
The acidification can be achieved using mineral acids, e.g., hydrochloric acid or sulphuric acid, or
lactic acid produced by microbiological fermentation [56]. Caseinates (sodium, potassium, or calcium
caseinate) can be obtained by adding alkalis (sodium hydroxide, potassium hydroxide, or calcium
hydroxide) to acid casein.
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3.2. Whey Protein Products
Whey products were initially viewed as a waste byproduct of cheese for thousands of years of
cheese history [57]. Whey can be basically categorized into acid whey and sweet whey [58]. Sweet
whey is the co-product of rennet cheese production, and the pH is usually around 6.0 or higher. Acid
whey comes from cottage cheese or Greek yogurt, and is formed where milk is acidified. Acid whey
has a pH below 5.0 and has a high lactic acid content; the direct processing of acid whey is limited [59],
and it is usually considered as the problematic stream. However, the recent Greek yogurt boom in the
USA has driven studies on acid whey utilization and processing [60–62], and acid whey is presently
repeating the success of sweet whey utilization.
Liquid sweet whey can be spray-dried into a powder. Sweet whey powder contains mostly lactose
(~80%) and protein (~10%). Membrane technology has been widely used for further processing of
whey products [63]. Whey protein concentrate (WPC) with a protein level up to 80% (e.g., WPC34 and
WPC80) can be produced using ultrafiltration (UF) [64]. Whey protein isolate (WPI) with a protein
content around 90%, can be achieved by combining with microfiltration (MF) in order to remove excess
fat [65]. The ash level can be further reduced by nano-filtration, ion-exchange, or resin to produce
deminilized whey protein products [66,67].
3.3. Membrane Filtered Milk Protein
Membrane filtration technology is able to separate milk protein from the serum phase, based on
the different sizes of molecules. Different membrane pore sizes result in different milk protein products.
Milk protein concentrate or isolate (MPC or MPI) is produced by ultrafiltration (UF) Both casein and
whey proteins are retained by the membrane, but lactose and minerals permeate [68]. Casein and
whey protein can be split by a microfiltration (MF) membrane, with casein being retained while whey
protein, along with lactose and minerals, can pass through the membrane, which results in micellar
casein products [69]. The whey stream can go through a process similar to the cheese whey process to
produce so-called milk serum protein concentrate and isolates [70].
4. Milk Protein Polymerization
Protein polymerization is essential for milk protein to be used as an adhesive. The casein micelle
itself is a heterogeneous polymer complex, made of different casein molecules. Additionally, casein
also interacts with whey protein to form casein–whey aggregates. The thiol group on the protruding
κ-casein tail can interact with β-Lg via thiol-disulphide interchange under heat treatment [71–73].
On the other hand, β-Lg can interact with α-La and BSA via the disulfide bonds [74,75]. Thus, a
polymer complex of casein and whey protein can be formed under heat treatment [76]. Figure 1
shows the interactions between casein and β-Lg; Figure 2 shows the protein interactions of whey
Polymers 2016, 8, 324
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protein molecules.
Figure
casein and
and β-Lg
β-Lginteractions.
interactions.
Figure 1.
1. Heat-induced casein
Polymers 2016, 8, 324
Figure 1. Heat-induced casein and β-Lg interactions.
5 of 12
Figure
Figure2.2.Whey
Wheyprotein
proteinpolymerization.
polymerization.
In order
strength
or or
fast
setting
properties,
protein
molecules
maymay
need
order to
toobtain
obtainaastrong
strongadhesive
adhesive
strength
fast
setting
properties,
protein
molecules
to be crosslinked.
ProteinProtein
crosslinking
can be achieved
by physical
(irradiation,
heat treatment,
etc.) [77],
need
to be crosslinked.
crosslinking
can be achieved
by physical
(irradiation,
heat treatment,
enzymatic
[78,79], or[78,79],
chemical
methods [80].
The strength
a protein
etc.)
[77], enzymatic
or (adding
chemicalcrosslinker)
(adding crosslinker)
methods
[80]. Theofstrength
ofnetwork
a proteinis
essential to
the bonding
strength.
Most
steps inMost
preparing
protein
polymer,
suchpolymer,
as heat treatment,
pH
network
is essential
to the
bonding
strength.
steps in
preparing
protein
such as heat
adjustment,pH
andadjustment,
the addition
of other
ingredients,
focus
on how to focus
increase
treatment,
and
the addition
of other
ingredients,
on protein
how to network
increase strength.
protein
Mixing
protein
polymers
with
chemical
crosslinkers
is
one
of
the
most
common
methods
to
cure
protein
network strength. Mixing protein polymers with chemical crosslinkers is one of the most common
adhesives.
Figures
3
and
4
demonstrate
examples
of
how
protein
is
crosslinked,
and
also
the
adhesion
methods to cure protein adhesives. Figures 3 and 4 demonstrate examples of how protein is crosslinked,
mechanisms
of wood mechanisms
and tissue of
adhesives.
Thermal
crosslinking
is widely
used inisplywood
and
also the adhesion
wood and
tissue adhesives.
Thermal
crosslinking
widely
manufacturing.
By
applying
both
heat
and
pressure,
protein
polymers,
especially
heat-sensitive
proteins,
used in plywood manufacturing. By applying both heat and pressure, protein polymers, especially
are denatured and
pressed
are able to
form
a veryand
firmare
adhesive
A ahot
press
is usually
used
for
heat-sensitive
proteins,
areand
denatured
and
pressed
able tofilm.
form
very
firm
adhesive
film.
Polymers
2016,
8, 324
6 of 12
plywood
manufacturing
in order to increase the bonding strength [81].
A hot press is usually used for plywood manufacturing in order to increase the bonding strength [81].
Figure 3. Protein crosslinked by polymeric methylene bisphenyl diisocyanate (PMDI) and the adhesion
Figure
3. Protein
crosslinked
by polymeric methylene bisphenyl diisocyanate (PMDI) and the adhesion
mechanisms
for wood
adhesive.
mechanisms for wood adhesive.
Figure 3. Protein crosslinked by polymeric methylene bisphenyl diisocyanate (PMDI) and the adhesion
6 of 12
mechanisms for wood adhesive.
Polymers 2016, 8, 324
Figure 4. Protein crosslinked by glutaraldehyde and the adhesion mechanism of biological glue.
Figure 4. Protein crosslinked by glutaraldehyde and the adhesion mechanism of biological glue. (A)
(A) glutaraldehyde; (B) protein polymer molecules; (C) tissue protein.
glutaraldehyde; (B) protein polymer molecules; (C) tissue protein.
Milk-Protein-Based
Adhesives
Applications
5.5.Milk-Protein-Based
Adhesives
Applications
5.1. Casein-Based Adhesives
5.1. Casein-Based Adhesives
The use of casein as glue products can be dated back to ancient Egypt [82]. The first US patent on
The use of casein as glue products can be dated back to ancient Egypt [82]. The first US patent on
using
casein products
products can
can be
be traced
traced back
back to
using casein
to the
the late-19th
late-19th century
century [83,84].
[83,84]. The
The early
early casein
casein adhesive
adhesive
preparation
usually
started
with
sour
milk
or
cheese
with
decanting
whey
to
remove
fat
and
water,
preparation usually started with sour milk or cheese with decanting whey to remove fat and water,
and
and
then
it
was
subjected
to
a
boiling
process
to
further
remove
water
and
to
denature
protein
[83,84].
then it was subjected to a boiling process to further remove water and to denature protein [83,84]. Other
Other ingredients,
such
like limewater,
alkali, limewater,
urea,always
were always
The micelle
casein micelle
ingredients,
such like
alkali,
or urea,orwere
added added
[83–86].[83–86].
The casein
is very
is very sensitive
to environmental
as pH
ion strength
[5,87].precipitation
Acid precipitation
is the
sensitive
to environmental
changes,changes,
such as such
pH and
ionand
strength
[5,87]. Acid
is the easiest
easiest
way
to
separate
casein
from
milk,
which
was
widely
used
in
the
early
days.
With
the
washed
way to separate casein from milk, which was widely used in the early days. With the washed and
and concentrated
acid casein
aggregates,
is usually
to solubilize
the casein
into viscous,
concentrated
acid casein
aggregates,
alkali alkali
is usually
addedadded
to solubilize
the casein
into viscous,
pastepaste-like
glue. Alkali
able
to reassemble
the micelle
structure
of casein
been
collapsed
like
glue. Alkali
pH is pH
ableis to
reassemble
the micelle
structure
of casein
thatthat
hashas
been
collapsed
by
by
acidification
[87,88].
Urea
and
ammonia
were
other
common
ingredients
for
casein
adhesives
in
the
acidification [87,88]. Urea and ammonia were other common ingredients for casein adhesives in the early
early days [85,89,90] to lower the viscosity by decreasing the H-bonds [91,92]. During an acid rinse,
CCPs are solubilized into the serum phase; thus, the micelle structure collapses, and, combined with a
heat process, casein can form a thick, insoluble, adhesive slurry, which resembles white glue. In most
cases, casein glues are available with a casein powder and an alkali, which are ready to be mixed in
water prior to use [82]. Many of the casein glues are generally no longer used due to the advent of
synthetic adhesive polymers; however, some are still in use [82]. In general, casein adhesive is easy to
process and provides good bond strength [82].
Casein-based adhesives are still used as a bottle label adhesive [82]. Bottle-labeling adhesive
for beer and soda bottles usually required ice-proof or water resistant properties, but also needed
to be easily washed off, since bottles may need to be returnable [93]. Acid-precipitated casein,
combined with metallic salts as crosslinkers, has been used for this purpose for about a century [86,94].
Casein-based wood glue was very common, due to its excellent water resistance, until synthetic resins
were invented [10,90]. The commercial products are usually sold as a dry powder, ready to be mixed
with water, right before use, because casein wood glue has a very short pot life. Some casein wood
adhesives are still in use today. One current commercial example is “Casein glue-N” from E Min Ta
An Casein Co. Ltd. (Xinjiang, China), which is used for furniture crossbanding and for beer labels.
5.2. Whey-Protein-Based Adhesives
A globular structure is not ideal for adhesive applications; therefore, treatments (such as heat and
solution polarity change) that denature the protein must be applied to globular proteins for adhesive
use [95]. Unlike the long history of casein adhesives, the use of whey protein for adhesives is relatively
new. Tschabold patented an adhesive from whey in 1953, which used condensed whey [96]. After that,
there was very little to be found in the literature until recent years.
Polymers 2016, 8, 324
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WPI has been used as a wood adhesive for both interior and exterior applications [97–101]. Similar
to casein wood adhesive, whey protein wood adhesive is also a ready-to-mix adhesive containing a
whey polymer aqueous solution and a crosslinker. The process usually starts with a high concentration
of WPI solution (20%–40%) polymerized at 60 ◦ C or higher, and is then combined with a synthetic
co-polymer, such as polyvinyl acetate (PVAC) and polyvinyl alcohol (PVA). The polymer suspension is
ready to be mixed with a crosslinker, such as phenol-formaldehyde oligomer or polymeric methylene
bisphenyl diisocyanate. Due to the thermo-setting properties of whey protein, desirable bonding
strength can also be obtained by hot pressing the whey protein polymer suspension without crosslinker
at 120–140 ◦ C [100].
Office or paper glue is another area where whey protein can be applied due to safety concerns [102].
Current commercial paper glues are commonly PVAC- and PVA-based. Polymerized whey protein
could be able to provide a desirable bonding strength, but the control of viscosity during storage,
to make the shelf life stable, is a challenge [103]. Polymerized whey protein suspension can be
combined with PVAC or PVA to obtain a good bonding strength, but the glue suspension is likely to gel
during the shelf life [103]; however, polymerized whey protein with polyvinyl pyrollidone (PVP) as a
co-binder has demonstrated very good bonding strength and a stable shelf life (at least two years) [103].
Carbohydrates (like sucrose) could be used with the polymerized whey protein suspension in order to
maintain the viscosity constant during storage [104]. Whey protein with PVP can also be used for glue
stick applications [102].
β-Lg, α-La, and BSA are all rich in lysyl residues, which have ε-amino groups [51,105,106].
The ε-amino group is very active and can react with crosslinkers, such as glutaraldehyde (GTA).
Globular protein and GTA adhesive has been commercially available for use as surgical glue, under
the brand BioGlue® . Tissue adhesive is a new alternate device for sutures, which could reduce physical
pain, prevent fluid leakage, an to shorten operation times [107,108]. BioGlue® is an FDA approved
tissue adhesive, which contains BSA as a protein polymer and GTA as a crosslinker. BSA is found in
both bovine blood and milk. Extensive in vivo evaluation of BioGlue® has been carried over the past
10 years [109–114]. The reaction mechanism of protein and GTA for use as tissue adhesive is depicted
in Figure 4. GTA can bridge the protein polymer and the tissue cells via reaction with the free amino
groups of the protein polymers and tissue cell proteins [108].
6. Summary
Synthetic petroleum-based resin will still dominate the adhesive industry due to its low cost
and excellent performance. However, as the rising concerns of environment pollution, human health,
and inert waste disposal, with respect to the use or production of synthetic adhesive polymers,
environmentally safe bio-resourced and renewable polymers still have good opportunities of use [115],
such as for paper glue for office/school/home use or in food packaging, wood adhesives, and biological
glues, where safety and other factors are more important than cost. Therefore, milk protein or other
natural polymer-based adhesives will have a niche in the market.
Conflicts of Interest: The authors declare no conflict of interest.
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